Candida albicans, as an opportunistic pathogen, can cause superficial and life-threatening candidiasis in immunocompromised individuals. The formation of surface-associated biofilms and the appearance of drug resistance pose a significant challenge for clinical intervention. In this study, a total of 104 hospital-acquired C. alibcans clinical isolates were collected from sterile sites and mucosal lesions of 92 infectious disease patients in the Shanghai Public Health Clinical Center and analysed. The resistance rates to fluconazole, itraconazole and voriconazole were 12.5 %, 15.4 % and 11.5 % respectively. Multilocus sequence typing (MLST) analysis identified 63 diploid sequence types (DSTs) with a decentralized phylogeny, of which 37 DSTs (58.7 %) had not been reported in the online MLST database. Loss of heterozygosity was observed in ACC1 and ADP1 sequences obtained from six sequential isolates from a patient receiving antifungal treatment, which exemplified the effect of microevolution on C. albicans genetic alterations. Biofilm formation capability, an important virulence trait of C. albicans, was variable among strains isolated from different anatomical sites (P50.0302) and affected by genotypes (P50.0185). The mRNA levels of the azole antifungal target ERG11 gene and efflux pump genes (CDR1, CDR2 and MDR1) were detected in 9-18.1 % of azole-resistant and susceptible-dose dependent (S-DD) isolates. Twelve mutations encoding distinct amino acid substitutions in ERG11 were found in azoleresistant and S-DD isolates. Among them, A114S, Y132H and Y257H substitution in the ERG11 gene may be primarily related to azole resistance. Taken together, we observed a high level of diversity within C. albicans isolates. Multiple inter-related underlying mechanisms, including genetic and environmental factors, may account for high surface adhesion or azole resistance in clinical C. albicans infections.Abbreviations: AAT1, aspartate aminotransferase; ACC1, Acetyl-coenzyme A carboxylase; ACT1, actin; ADP1, ATP-dependent permease; ALS, hyphaspecific surface protein; BSI, nosocomial bloodstream infection; CDR1, CDR2, ATP-dependent efflux pumps; CLSI, Clinical and Laboratory Standard Institute; DST, diploid sequence type; ERG11, cytochrome P450 14a-demethylase; HWP1, hypha-specific cell wall protein; MDR1, major facilitator pump; MLST, multilocus sequence typing; MPI, mannose phosphate isomerase; SAP, secreted aspartyl protease; S-DD, susceptible-dose dependent; SYA1, alanyl-RNA synthetase; VPS13, vacuolar protein sorting protein; XTT, 2,3-bis-(2-methoxy-4-nitro-5-sulphenyl)-2H-tetrazolium-5-carboxanilide; YPD, yeast extract, peptone, dextrose medium; ZWF1b, glucose-6-phosphate dehydrogenase.
AimsTo investigate the histopathological manifestations of two fatal cases of H7N9 influenza A virus infection.MethodsPulmonary and hepatic specimens from two fatal cases of H7N9 influenza virus infection were examined using H&E staining. Additionally, in situ hybridisation was performed with probes (ViewRNA) targeting H7N9 RNA and IP-10, interleukin (IL)-6 mRNA. The distribution of surfactant protein A (SP-A), surfactant protein B (SP-B), CD3, CD4, CD8, CD68 and C4d were determined with immunohistochemistry.Results Apart from the typical diffuse alveolar damage and hyaline membrane observed in severe influenza infection, we detected H7N9 RNA and massive intrapulmonary production of IP-10 and IL-6 mRNA using in situ hybridisation. Hyperplasia of type II pneumocytes was observed by H&E staining and immunohistochemistry. Proliferating macrophages and clustered neutrophils in the infected lungs were observed, whereas T lymphocytes, especially CD4T helper cells, were markedly depleted. No obvious complement deposition was found in lung tissues.Conclusions Our findings suggest that H7N9 influenza virus induced an immunological response towards overt pulmonary inflammation and systemic lymphopenia which led to intense alveolar damage and respiratory failure.
Metagenomic next-generation sequencing (mNGS) holds promise as a diagnostic tool for unbiased pathogen identification and precision medicine. However, its medical utility depends largely on assay simplicity and reproducibility. In the current study, we aimed to develop a streamlined Illumina and Oxford Nanopore-based DNA/RNA library preparation protocol and rapid data analysis pipeline. The Illumina sequencing-based mNGS method was first developed and evaluated using a set of samples with known aetiology. Its sensitivity for RNA viruses (influenza A, H1N1) was < 6.4 × 102 EID50/mL, and a good correlation between viral loads and mapped reads was observed. Then, the rapid turnaround time of Nanopore sequencing was tested by sequencing influenza A virus and adenoviruses. Furthermore, 11 respiratory swabs or sputum samples pre-tested for a panel of pathogens were analysed, and the pathogens identified by Illumina sequencing showed 81.8% concordance with qPCR results. Additional sequencing of cerebrospinal fluid (CSF) samples from HIV-1-positive patients with meningitis/encephalitis detected HIV-1 RNA and Toxoplasma gondii sequences. In conclusion, we have developed a simplified protocol that realizes efficient metagenomic sequencing of a variety of clinical samples and pathogen identification in a clinically meaningful time frame.
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